This application is for funds to continue studies on the biogenesis of adrenal chromaffin vesicles. During the past years we have demonstrated the incorporation of 3H-leucine into soluble and membrane-bound dopamine-Beta-hydroxylase and into the soluble chromogranins of the chromaffin vesicles. During the proposed grant period we plan to consolidate and extend these findings using both in vitro and in vivo preparations. The objectives of these investigations are to determine whether chromaffin vesicle membranes are recycled during reformation of chromaffin vesicles or whether vesicle membranes are formed de novo, and to obtain information on the assembly of the vesicle membrane. Studies will be carried out to determine whether all the soluble protein constituents of the vesicles are synthesized and incorporated into chromaffin vesicles in parallel, and the relationship between the soluble and membrane bound form of dopamine-Beta-hydroxylase. Using in vivo preparations, studies on the biosynthesis of soluble and membrane bound dopamine-Beta-hydroxylase and chromogranins and their incorporation into chromaffin vesicles will be carried out under resting conditions and during and after physiological stimulation. In the in vitro system a component of the cytoplasmic proteins is rapidly and highly labeled with 3H-leucine. Attempts will be made to isolate and characterize this protein.